Methods and compositions for determining the presence or absence of dna aberrations

ABSTRACT

The invention consists of methods and compositions for detecting the presence or absence of a DNA aberration by analyzing fluorescence emission characteristics in sperm cells or sperm nuclei, which generally consists of entraining sperm cells or sperm nuclei stained with a DNA selective dye in sheath fluid; exposing the entrained sperm cells or sperm nuclei to electromagnetic radiation; determining a forward fluorescence characteristic and a side fluorescence characteristic of individual events associated with the exposed sperm cells or sperm nuclei; and gating the individual events based on the forward fluorescence characteristic and the side fluorescence characteristic with a criterion.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States Provisional PatentApplication No. 62/655,040 filed Apr. 9, 2018 and U.S. ProvisionalPatent Application No. 62/673,668 filed May 18, 2018. The entiredisclosures of which are incorporated herein by reference.

BACKGROUND

In mammals, the male (sire) haploid cells (sperm) are presented byejaculation in very large numbers comprising millions or even billionsof cells, each of which carry a somewhat random distribution ofautosomes (normally one of a pair of equal sized autosomes) as well assex chromosomes (X and Y) that normally distribute ½ of the totalhaplotype of the male into each sperm. In normal healthy males, thesperm population is typically two distinct populations, where variationin total DNA content is based only on the sex chromosome and sorting ofsuch populations of sperm by flow cytometry is a well-established andnon-invasive method to preselect sex (create percent of bias of male orfemale) in mammals.

The use of genetic analysis of individual breeding animals by DNAgenotyping or DNA sequencing methods has become a low-cost method toanalyze the genetic microstructure and mathematical distribution ofhaplotype inheritance and is used in large scale to determine thebreeding value of an animal. Large stable DNA macrostructuralaberrations such as translocations are not analyzed by theabove-mentioned microstructural DNA analysis methods, but since they canalso lead to poor reproductive performance, such as reduction in littersize (in multiparous species) or pregnancy rates (in uniparous species)by mechanism of early embryonic death, low-cost methods to screen forthem are needed.

A common problem, for example in swine breeding, is created whentranslocations or other stable DNA aberrations are carried forward,particularly in breeding males, by non-lethal inheritance. When thenegative phenotype is litter size, the reliance on that phenotypicalobservation to cull out breeding boars and sows is problematic becauseprogeny carriers are created, and if these animals are then used togenerate new breeding males and females they may also be carriers andthe problem can remain or even be amplified. For this reason, it hasbecome a common practice to use the long-standing method of karyotypeanalysis to screen breeding animals, in special boars, prior to theiruse. Karyotype analysis has the advantage that it can commonly identifylarge stable changes in DNA macrostructure by reliable and well definedmethods, and the advent of computer assisted image analysis has madekaryotype analysis routine. Nonetheless, karyotype analysis typicallyrequires handling of fresh specimens (mainly blood), the cultivation ofclonal cell lines in defined but artificial media and the analysis ofdiploid somatic cells as a proxy to direct analysis of germ line cells.The process typically takes a minimum of several days, if not even weeksto complete. It is not highly statistically significant, as the numberof cells analyzed is small. There are also limitations in the thresholdof percent of DNA change that can be determined. Furthermore, karyotypeanalysis does not measure any abnormal effects in spermatogenesis thatmight lead to materially important changes in DNA distribution withinsperm populations (gamete aneuploidy).

Additionally, in species such as bovines where artificial inseminationis used extensively but conception rate information is difficult andtime consuming to recover, karyotype analysis is not reliable andlow-cost enough to be used industrially. The current karyotypingprocedures takes approximately 3 weeks. A sample must be shipped byovernight courier and in some cases shipments are delayed, and thesample is lost and unable to be analyzed requiring resampling.Karyotyping is costly and prices are expected to increase with a lack ofsufficient facilities to perform karyotyping, with some facilitiesdiscontinuing service.

Translocations happen at a rate of 1% in swine—therefore a very highnumber of tests are normal at a high cost. The effect of a translocatedboar's semen is a loss of about 4-5 piglets per litter. One single boarcan produce thousands of pigs in a lifetime, so if those litter sizesare smaller there is lost productivity. Because of the problem, which isthat approximately ⅓ of the sperm produced by a translocated boar willbe non-viable due to reduced amount of DNA, decreased fecundity occurs.

SUMMARY OF THE INVENTION

One embodiment of the invention encompasses a method of analyzingfluorescence emission characteristics in sperm cells or sperm nuclei(i.e., sperm heads), comprising entraining sperm cells or sperm nucleistained with a DNA selective dye in sheath fluid; exposing the entrainedsperm cells or sperm nuclei to electromagnetic radiation; determining aforward fluorescence characteristic and a side fluorescencecharacteristic of individual events associated with the exposed spermcells or sperm nuclei; gating the individual events based on the forwardfluorescence characteristic and the side fluorescence characteristicwith a criterion; and determining the presence or absence of a DNAaberration from the gated individual events. In a further embodiment,the DNA selective dye is Hoechst 33342. Another embodiment furthercomprises the step of orienting the entrained sperm cells or spermnuclei. In another embodiment, the step of exposing the sperm cells orsperm nuclei to electromagnetic radiation comprises exposing the spermcells or sperm nuclei to a laser beam with modified beam profile. In yetanother embodiment, the criterion encompasses a subpopulation oforiented sperm cells or sperm nuclei. In these embodiments, the spermmay comprise sperm cells or sperm nuclei from a first and a secondmammalian species. In a further embodiment, determining the presence ofa DNA aberration from the gated individual events comprises detectingmore than two peaks or modes, or a peak to valley ratio of 80% or less,70% or less, 60% or less, or 50% or less, on a histogram of fluorescenceintensities, or measureable differences in co-efficient of variation intwo or more modes. In a yet further embodiment, determining the presenceof a DNA aberration from the gated individual events comprises detectinga difference between a peak to valley ratio on a histogram offluorescence intensities of sperm cells or sperm nuclei from the firstmammalian species and a peak to valley ratio on a histogram offluorescence intensities of sperm cells or sperm nuclei from the secondmammalian species or measureable differences in co-efficient ofvariation between two or more modes created by the first and secondmammalian species. Another embodiment of the invention comprisessubjecting the entrained sperm cells or sperm nuclei to orientinghydrodynamic forces such as those imparted by an orienting nozzle.

The invention also encompasses a method of analyzing fluorescenceemission characteristics in sperm cells or sperm nuclei, comprisingentraining sperm cells or sperm nuclei stained with a DNA selective dyein sheath fluid; exposing the entrained sperm cells or sperm nuclei toelectromagnetic radiation; determining two or more fluorescence emissioncharacteristics of individual events associated with the exposed spermcells or sperm nuclei; gating the individual events based on the two ormore fluorescence emission characteristics with a first criterion forfurther processing; gating the further processed individual events witha second criterion; and determining the presence or absence of a DNAaberration from the twice gated individual events. In a furtherembodiment, the first criterion encompasses a first subpopulation oforiented sperm cells or sperm nuclei. In a yet further embodiment, thesecond criterion encompasses a second subpopulation of sperm cells orsperm nuclei comprising 25% to 75% of the first subpopulation. In aneven further embodiment, the step of determining the presence or absenceof DNA aberrations in the sperm cells or sperm nuclei from the twicegated individual events further comprises evaluating a thirdsubpopulation of sperm cells or sperm nuclei within the firstsubpopulation, wherein the third subpopulation of sperm cells or spermnuclei excludes the second subpopulation of sperm cells or sperm nuclei.In an additional embodiment, the second criterion encompasses 25% to 75%of sperm around a median value of a fluorescence emissioncharacteristic. In a further embodiment, the step of determining thepresence or absence of a DNA aberration in the sperm cells or spermnuclei from the twice gated individual events further comprisesdetermining the quantity of peaks or modes in a twice gated fluorescenceemission characteristic. In an even further embodiment, the step ofdetermining the presence or absence of a DNA aberration in the spermcells or sperm nuclei from the twice gated individual events furthercomprises analyzing a coefficient of variation of a fluorescenceemission characteristic associated with the first subpopulation of spermcells or sperm nuclei or the second subpopulation of sperm cells orsperm nuclei. In an additional embodiment, the step of determining thepresence or absence of a DNA aberration in the sperm cells or spermnuclei from the twice gated individual events further comprisesgenerating a first univariate plot based on a fluorescence emissioncharacteristic of the first subpopulation or generating and a secondunivariate plot based on a fluorescence emission characteristic of thesecond subpopulation. In a further embodiment, the step of determiningthe presence or absence of a DNA aberration in the sperm cells or spermnuclei from the twice gated individual events further comprisesanalyzing a peak to valley ratio of the first univariate plot or a peakto valley ratio of the second univariate plot or measureable differencesin co-efficient of variation between modes in multivariate plots. In aneven further embodiment, the method further comprises separating tailsor midpieces from the sperm nuclei by centrifugation. In a furtherembodiment, the DNA selective dye is Hoechst 33342. Another embodimentfurther comprises the step of orienting the entrained sperm cells orsperm nuclei. In another embodiment, the step of exposing the spermcells or sperm nuclei to electromagnetic radiation comprises exposingthe sperm cells or sperm nuclei to a laser beam with modified beamprofile. In these embodiments, the sperm cells or sperm nuclei maycomprise sperm cells or sperm nuclei from a first and a second mammalianspecies. In a further embodiment, determining the presence of a DNAaberration comprises detecting more than two peaks or modes, or a peakto valley ratio of 80% or less, 70% or less, 60% or less, or 50% orless, on a histogram of fluorescence intensities or measureabledifferences in co-efficient of variation between modes in multivariateplots. In a yet further embodiment, determining the presence of a DNAaberration comprises detecting a difference between a peak to valleyratio on a histogram of fluorescence intensities of sperm cells or spermnuclei from the first mammalian species and a peak to valley ratio orco-efficient of variation in modes on a histogram of fluorescenceintensities of sperm cells or sperm nuclei from the second mammalianspecies. Another embodiment of the invention comprises subjecting theentrained sperm cells or sperm nuclei to orienting hydrodynamic forcessuch as those imparted by an orienting nozzle.

The invention is also embodied by a method of analyzing a fluorescenceemission characteristic in sperm cells or sperm nuclei, comprisingstaining the sperm cells or sperm nuclei with a DNA selective dye;entraining the stained sperm cells or sperm nuclei in sheath fluid;exposing the entrained sperm cells or sperm nuclei to electromagneticradiation; determining a fluorescence emission characteristic of theexposed sperm cells or sperm nuclei; generating a first multivariateplot based on the fluorescence emission characteristic; providing afirst gate on the first multivariate plot; generating a secondmultivariate plot based on the first gate; providing a first sort regionon the second multivariate plot encompassing a first subpopulation ofsperm and a second sort region on the second multivariate plotencompassing a second subpopulation of sperm; separating the firstsubpopulation of sperm cells or sperm nuclei and the secondsubpopulation of sperm cells or sperm nuclei; generating a firstunivariate plot based on a fluorescence emission characteristic of theseparated first subpopulation of sperm cells or sperm nuclei; generatinga second univariate plot based on a fluorescence emission characteristicof the separated second subpopulation of sperm cells or sperm nuclei;and determining the presence or absence of a DNA aberration based on thefirst univariate plot or the second univariate plot. In anotherembodiment, the method further comprises the step of separating tails ormidpieces from the sperm nuclei by centrifugation. In yet anotherembodiment, the first subpopulation of sperm cells or sperm nucleicomprises 25% to 75% of the sperm population. In an even furtherembodiment, the second subpopulation of sperm cells or sperm nucleiexcludes the first subpopulation of sperm cells or sperm nuclei. In anadditional embodiment, the first sort region encompasses the center ofthe second multivariate plot. In yet another embodiment, the methodcomprises the step of determining the quantity of peaks or modes of thefirst univariate plot or the second univariate plot, analyzing acoefficient of variation of the first univariate plot or the secondunivariate plot, or analyzing a peak to valley ratio of the firstunivariate plot or the second univariate plot. In a further embodiment,the DNA selective dye is Hoechst 33342. Another embodiment furthercomprises the step of orienting the entrained sperm cells or spermnuclei. In another embodiment, the step of exposing the sperm cells orsperm nuclei to electromagnetic radiation comprises exposing the spermcells or sperm nuclei to a laser beam with modified beam profile. Inthese embodiments, the sperm cells or sperm nuclei may comprise spermcells or sperm nuclei from a first and a second mammalian species—insuch embodiments, the first subpopulation of sperm cells or sperm nucleicomprises 25% to 75% of the sperm cells or sperm nuclei from the firstmammalian species. In a further embodiment, determining the presence ofa DNA aberration comprises detecting more than two peaks or modes, or apeak to valley ratio of 80% or less, 70% or less, 60% or less, or 50% orless, on a histogram of fluorescence intensities or measureabledifferences in co-efficient of variation between modes in multivariateplots. Another embodiment of the invention comprises subjecting theentrained sperm cells or sperm nuclei to orienting hydrodynamic forcessuch as those imparted by an orienting nozzle.

Another embodiment of the invention comprises a method of analyzing afluorescence emission characteristic in sperm cells or sperm nuclei,comprising staining the sperm cells or sperm nuclei with a DNA selectivedye; entraining the stained sperm cells or sperm nuclei in sheath fluid;exposing the entrained sperm cells or sperm nuclei to electromagneticradiation; determining a fluorescence emission characteristic of theexposed sperm cells or sperm nuclei; generating a first multivariateplot based on the fluorescence emission characteristic; providing afirst gate on the first multivariate plot; generating a secondmultivariate plot based on the first gate; providing a first sort regionon the second multivariate plot encompassing a first subpopulation ofsperm cells or sperm nuclei and a second sort region on the secondmultivariate plot encompassing a second subpopulation of sperm cells orsperm nuclei, wherein the first sort region encompasses the center ofthe second multivariate plot. In a further embodiment, the firstsubpopulation of sperm cells or sperm nuclei comprises 25% to 75% of thesperm cell or sperm nuclei population. In a yet further embodiment, thesecond subpopulation of sperm cells or sperm nuclei excludes the firstsubpopulation of sperm cells or sperm nuclei. Another embodiment of theinvention comprises subjecting the entrained sperm cells or sperm nucleito orienting hydrodynamic forces such as those imparted by an orientingnozzle.

In a yet further embodiment, determining the presence of a DNAaberration comprises detecting a difference between a peak to valleyratio on a histogram of fluorescence intensities of sperm cells or spermnuclei from the first mammalian species and a peak to valley ratio orco-efficient of variation in modes on a histogram of fluorescenceintensities of sperm cells or sperm nuclei from the second mammalianspecies.

Another embodiment of the invention comprises a composition comprisingsonicated sperm cells or sperm nuclei, a DNA selective dye, anaggregation reducing compound and a buffer. In a further embodiment, theaggregation reducing compound comprises egg yolk, iodixanol, lecithin,bovine serum albumin, gelatin, collagen or hydrolyzed collagen,arabinogalactan, or a chemically defined polyethylene or polypropyleneglycol. In a yet further embodiment, the sonicated sperm cells or spermnuclei comprise sperm cells or sperm nuclei from a first non-humanmammalian species and sperm cells or sperm nuclei from a secondnon-human mammalian species. In a further embodiment, the DNA selectivedye is Hoechst 33342.

In a further embodiment of the invention, any of the methods fordetermining the presence or absence of a DNA aberration disclosed hereincan further comprise the step of culling the male from whom the spermcells or sperm nuclei are obtained or derived based on the determinedpresence of a DNA aberration in the male. In another embodiment of theinvention, any of the methods for determining the presence or absence ofa DNA aberration disclosed herein can further comprise the step removingthe male, from whom the sperm cells or sperm nuclei are obtained orderived, from a breeding program in a genetic nucleus or a herd based onthe determined presence of a DNA aberration in the male. In anotherembodiment, the method further comprises karyotyping the male from whomthe sperm cells or sperm nuclei are obtained or derived based on thedetermined presence of a DNA aberration in the male. In an additionalembodiment, the method further comprises karyotyping the dam of the malefrom whom the sperm cells or sperm nuclei are obtained or derived basedon the determined presence of a DNA aberration in the male. In anadditional embodiment, the method further comprises culling the dam ofthe male from whom the sperm cells or sperm nuclei are obtained orderived based on the determined presence of a DNA aberration in the maleor an abnormal karyotype in the dam. In a further embodiment, the methodfurther comprises determining the presence or absence of a DNAaberration (using any of the methods disclosed herein) in, orkaryotyping, the progeny of the dam of the male from whom the spermcells or sperm nuclei are obtained or derived based on the determinedpresence of a DNA aberration in the male. In a further embodiment, themethod further comprises culling progeny of the dam of the male fromwhom the sperm cells or sperm nuclei are obtained based on thedetermined presence of a DNA aberration or abnormal karyotype in theprogeny or the determined presence of a DNA aberration in the male.

It should be understood that any of the embodiments disclosed herein fordetermining the presence or absence of a DNA aberration can be employedto detect the presence or absence of the aberration in either spermcells or sperm nuclei (i.e., sperm with their tails and midpiecesremoved, by for example, sonication)—that is, either sperm or spermnuclei can be analyzed with the methods of the invention.

The invention also encompasses an improved method for making spermnuclei comprising combining a DNA-selective dye and an aggregationreducing compound with a sperm cell sample to create a sperm cellmixture; and sonicating the sperm cell mixture to create stained spermnuclei.

Another embodiment of the invention comprises a composition comprisingunsorted sperm nuclei, an aggregation-reducing compound and a DNAselective dye, wherein the composition has been sonicated. In a furtherembodiment, the DNA selective dye is Hoechst 33342. In anotherembodiment, the sperm nuclei are derived from sperm cells from one male.In another embodiment, the composition has a temperature of 45° C. orgreater. In a particular embodiment, the aggregation-reducing compoundis egg yolk.

An additional embodiment of the invention comprises a method ofprocessing sperm cells comprising providing an unsorted sperm cellsample; combining a DNA selective dye and an aggregation-reducingcompound with the unsorted sperm cell sample to create a sperm cellmixture; and sonicating the sperm cell mixture to create stained spermnuclei. In another embodiment, the DNA selective dye is Hoechst 33342.In a yet further embodiment, the unsorted sperm cell sample is obtainedfrom one male. In another embodiment, the sperm cell mixture has atemperature of 45° C. or greater during the step of sonication. In a yetfurther embodiment, the aggregation-reducing compound is egg yolk. In anadditional embodiment, the method is completed in 20 minutes or less.

The improved sperm nuclei of the invention can be used in any of theaforementioned methods of determining the presence or absence of a DNAaberration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of multivariate and univariate plots as well asgates used for logical gating of abnormal boar sperm nuclei (i.e.,comprising a DNA aberration).

FIG. 2 illustrates part of a flow cytometer used to analyze and thensort a sperm composition to form one or more subpopulations.

FIG. 3 illustrates a microfluidic chip used to analyze and then sort asperm composition.

FIG. 4 illustrates a univariate plot in the form of a histogramgenerated by an analyzer or sorter.

FIGS. 5-11 are flow cytometer images showing exemplary histograms forthe five different categories of stable chromosomal translocation foundin boars, produced using the parse sorting method of the invention.

FIGS. 12-18 are flow cytometer images showing histograms for boar spermnuclei analyzed using the parse sorting method of the invention.

FIGS. 19-22 are flow cytometer images showing histograms for boar spermnuclei analyzed using the logical gating method of the invention.

FIGS. 23-33 are flow cytometer images showing histograms for boar spermnuclei analyzed using the parse sorting method of the invention.

FIG. 34 shows flow cytometer images showing histograms for bull spermnuclei analyzed using the parse sorting method of the invention.

FIG. 35 shows a screenshot from a flow cytometer showing multivariateplots and a univariate plot of forward fluorescence intensities of astained sperm cell sample indicating the presence of a DNA aberration(Category IV translocation) in a boar.

FIG. 36 shows a screenshot from a flow cytometer showing multivariateplots and a univariate plot of forward fluorescence intensities of astained sperm cell sample indicating the presence of a DNA aberration(Category III translocation) in a boar.

FIG. 37 shows a screenshot from a flow cytometer showing multivariateplots and a univariate plot of forward fluorescence intensities of astained sperm cell sample indicating the presence of a DNA aberration(Category IV translocation) in a boar.

FIG. 38 shows a flow cytometer image showing a histogram for asubpopulation from the center sort region indicating the presence of aDNA aberration in a bull.

FIG. 39 shows a flow cytometer image showing a histogram for asubpopulation from the flanking sort region indicating the presence of aDNA aberration in a bull.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention encompasses a method that uses alow-cost and readily available biomaterial in sperm, facilitates therapid analysis of said sperm (or derived sperm nuclei) in a matter ofminutes (in contrast to days or weeks,) and depending on the level ofprecision and accuracy of the equipment and operator, can generatestatistically significant proof and quantification of changes in totalDNA that may be as low as 0.1%. Parallelization of the invention insample handling and total per-sample analysis time of less than one hourcan facilitate the analysis of about 10 samples or more per day andrelatedly as many as 2500 samples per year at costs per sample that maybe as low as 20% of karyotype analysis costs. The invention is alsocapable of yielding a more sensitive measurement of the size of DNAaberrations than is typical with karyotyping. Using the invention,identity mapping to show identical genotypes (related carrier animals)is facilitated, and the study of the way such aberrations arise andinherit is also facilitated. It should be understood that the inventionin any of its embodiments can be used to determine the presence orabsence of a DNA aberration using either sperm or sperm nuclei.

Although the invention may substitute or replace higher cost and timeconsuming karyotyping, it is also complementary, as the measured featureis distribution of total DNA in different subpopulations withoutspecification of which chromosomes are affected. In some embodiments,the invention can be performed using state-of-the-art equipment used inthe sex-selection of mammals in industrial scale and can therefore beintroduced to existing users of sex-selection technology with limitednew capital investment.

Since in some embodiments, the invention can use frozen sperm and infurther embodiments be micro-scaled to use as few as 50 million sperm,it can be performed using one or more cryopreserved semen straws, evenfrom animals that are not living. In this way, even though in someembodiments the invention may be concentrated at locations thatspecialize in sex-selection in general, the shipment and contractanalysis of samples will be possible.

Since the invention uses flow cytometry, future advances inminiaturization, automation and sensitivity of sperm analysis by flowcytometry will facilitate future reduction in cost of using theinvention and parallelization of analysis, if needed. As used herein,the term “flow cytometry” also encompasses microfluidics, and the term“flow cytometers” encompasses both flow cytometers and microfluidicdevices generally. Certain embodiments of the invention may furthercomprise the use of kits, where ready-to-use reagents and consumablematerials can allow the dissemination of the method to large numbers ofdifferent locations with same quality of analysis.

In certain embodiments, the invention does not require the use ofcomputational analysis of histogramatic data produced in flow cytometry(as exemplified in flow cytometry standard [“FCS”] files), but sincesuch data can easily be produced during the analysis, the precision withwhich data can be analyzed can be improved by using a large number ofalready existing software packages for analyzing flow cytometry data. Inother embodiments, computational analysis of the data, includinghistogramatic data, produced by the invention may be utilized.

It is contemplated that the invention can be applied to many hundreds ofthousands or millions of breeding sires (e.g., boars and bulls) and canreadily become a standard and essential method in the screening ofbreeding value and reproductive performance of livestock or otheranimals, including humans. Additionally, although methods for artificialchromosomes (synthetic autosomes) are still emerging, cattle and swinegeneticists are developing and utilizing safe and effective methods ofartificial transgenesis. In certain embodiments, the analysis ofstability and integrity of heritability of large DNA transgenes thatincrease the total DNA content may be analyzed or studied by theinvention, and it is contemplated that the use of artificial transgenesmay be facilitated by sorting methods similar to the sex-selectionmethods used today.

Generally, in one aspect of the invention, a fresh or frozen ejaculateof sperm is the raw material to be processed. A DNA selective dye orstain (including, but not limited to, Hoechst 33342) is combined in aliquid media with appropriate pH, modifying buffers and aggregationreducing chemicals. The sperm mixture is then optionally sonicated tosimultaneously provide heat that accelerates the rate of stainpenetration and saturation and to disrupt sperm cell structure to removetails and midpieces, thereby creating stained sperm nuclei.

One embodiment of the invention comprises entraining the stained spermor sperm nuclei in sheath fluid and exposing the entrained sperm orsperm nuclei to electromagnetic radiation. In connection with individualevents associated with the exposed sperm cells or sperm nuclei, theinvention further comprises determining a forward fluorescencecharacteristic, which in certain embodiments correlates to the quantityof DNA in a sperm cell or a sperm nuclei and a side fluorescencecharacteristic, which in certain embodiments correlates to theorientation of a sperm cell or sperm nuclei. Next, the inventioncontemplates gating the individual events based on the forwardfluorescence characteristic and side fluorescence characteristic withone or more criterion, e.g., oriented sperm or sperm nuclei, or DNAcontent. In certain embodiments of the invention, one can determine thepresence or absence of a DNA aberration from the gated individualevents. “Chromosomal aberration” or “DNA aberration” as used hereinencompasses a chromosomal translocation, a transgene, or any other statein which the DNA content of sperm from an individual varies from cell tocell other than variation attributable to sex chromosomes. For example,a univariate plot can be generated based on forward fluorescenceintensity of the gated individual events. Typically, in normal mammaliansperm or sperm nuclei, the univariate plot would show two distinct peaks(each peak corresponding to X-bearing or Y-bearing sperm). In anindividual with a chromosomal or DNA aberration, however, the univariateplot may show more than two peaks. Additionally, in certain embodimentsof the invention, a peak to valley ratio is calculated for the peaksshown on the univariate plot, with a peak to value ratio of 50% or lessindicating the presence of a chromosomal aberration, for example. Inrelated embodiments, comparison of co-efficient of variation between twoor more peaks or modes using mathematical analysis (Gaussian or thelike) from histogramatic data is used.

In a further embodiment of the invention, the sperm cells or spermnuclei are analyzed using a “logical gating” method using a flowcytometer or microfluidic device. This embodiment comprises gating asubpopulation of oriented sperm or sperm nuclei on a multivariate plotwith a first gate and then generating a second multivariate plot basedon the first gate. The method further comprises creating a first “centergate” on the second multivariate plot that selects a percentage of spermcells or sperm nuclei distributed near and around the mean of populationdistribution (two or more “peaks”), and a second “flanking gate” on thesecond multivariate plot that selects for the remaining sperm cells orsperm nuclei outside of the center gate or a substantial portionthereof. A univariate plot can be generated based on forwardfluorescence intensity for each of the center gate and the flankinggate. Normal sperm cell or nuclei samples that comprise two peaks(normal X and normal Y) result in center and flanked populations thatshow the same two peaks on each of the univariate plots with similarpeak to valley ratios, typically greater than 50%. In contrast, abnormalsperm cell or nuclei samples that contain smaller or larger autosomes orsets of autosomes will result in differences between the number of peaksthat appear in the center and flanked populations or peak to valleyratios of 50% or less, for example.

FIG. 1 shows examples of multivariate and univariate plots as well asgates used for logical gating of abnormal boar sperm nuclei (i.e.,comprising a DNA aberration). Plot 1 represents a multivariate plotshowing forward fluorescence along the y-axis and side fluorescencealong the x-axis. A gate 5 has been placed around events that areoriented well enough to capture optimal resolution. Each dot representsan event detected by the flow cytometer. Events outside of gate 5 arelow resolution or unresolvable. The events within gate 5 are thenplotted on plot 2, which shows forward fluorescence along the y axis andan integral of forward fluorescence along the x-axis. Essentially, plot2 depicts the gated events in plot 1 rotated approximately 90° towardsthe viewer. In plot 2, a center gate 7 and a flanking gate 9 are placed.The events within flanking gate 9 are then plotted on plot 3, whichshows forward fluorescence intensity along the y axis and an integral offorward fluorescence intensity along the x-axis, and plot 4, which is ahistogram showing the forward fluorescence intensities of events gatedby the flanking gate 9. The events within center gate 7 are plotted onplot 5, which shows forward fluorescence intensity along the y axis andan integration of forward fluorescence intensity along the x-axis, andplot 6, which is a histogram showing the forward fluorescenceintensities of events gated by the center gate 7. As can be seen in FIG.1, the histogram (plot 4) generated by events in the flanking gate 7shows an abnormal number of modes, or peaks (normal being two modes, orpeaks, in mammalian species) and corresponds to a boar that has achromosomal translocation.

In another embodiment of the invention, sperm cells or sperm nuclei areactually “parse sorted” using a flow cytometer or microfluidic device,where “parse sorting” encompasses sorting a first “center sort region,”selecting for a percentage of sperm cells or sperm nuclei distributednear and around the mean of population distribution (two or more“peaks”), and a second “flanking sort region,” which typically selectsfor the remaining sperm cells or sperm nuclei outside of the center sortregion. In some embodiments, more than two subpopulations may be sorted,for example, a center sort region and two separate flanking sortregions. The separate, parse sorted sperm cell or sperm nucleisubpopulations are then analyzed and compared. Normal sperm cell ornuclei samples that comprise two peaks (normal X and normal Y) result incenter and flanking populations that show the same two peaks. Abnormalsamples that contain smaller or larger autosomes or sets of autosomeswill result in differences in the number and relative DNA content ofpeaks that appear in the center and flanking populations.

In a further embodiment of parse sorting, the separate populations thatare sorted may include further dividing the flanking sort region toseparate the high-total-DNA content sperm cells or sperm nuclei on oneside of the flank from the low-total-DNA content sperm cells or spermnuclei on the other side of the flank, while also separating apopulation from the center where average-total-DNA content sperm cellsor sperm nuclei are selected.

In a further embodiment of the invention, sperm cells or sperm nuclei ofa second species may be added to the sperm cell or nuclei sample toprovide an internal standard to establish a reference of known particlecount, known total fluorescence intensity, known percent DNAdifferential between sex chromosomes, known peak to value ratio, orreference standard deviation of Gaussian distribution. In certainembodiments, a method for creating the internal standard comprises theaddition of sperm or sperm nuclei of a known quantity from a secondarymammalian species to a staining mixture comprising a DNA selective dyewith appropriate conditions of sonication, which can then be added as aninternal standard to the sperm cell or nuclei sample of the firstmammalian species that is to be analyzed for chromosomal aberrations.For example, FIG. 9 shows histograms for parsed center and parsed flanksubpopulations of sperm nuclei from a boar that includes an internalstandard comprising bovine sperm nuclei. The histogram for the parsedcenter subpopulation in FIG. 9 clearly indicates the presence of a DNAaberration in the boar's sperm nuclei since the peak to valley ratiosfor that subpopulation is significantly lower than the peak to valleyratio shown on the histogram of the internal standard.

In one embodiment, mathematical analysis of the Gaussian distributionsof normal and abnormal peaks within single samples and across groups ofsamples can be used to make a differential determination of normal vsabnormal (i.e., the presence or absence of a DNA aberration) moresensitive and precise.

Generally, the invention is rapid. However, certain embodiments of theinvention may take longer than other embodiments. For example, in someembodiments, sperm nuclei populations may be partially or fully purifiedaway from tails and midpieces prior to staining. Additionally, incertain embodiments, staining may be performed at temperatures lowerthan in some embodiments of the invention. In some embodiments, theinvention can use slower and more benign sample handling methods ifmaintaining the viability of sperm is required, such as foregoingsonication.

Some embodiments of the invention may specify a target temperature atwhich sonication is completed/terminated. The target temperature can be5° C., 45° C., or 70° C., or can be in the range between 5-70° C.,30-70° C., or 55-65° C. In some embodiments of the invention the targettemperature may be reached in a time between 1 minute and 5 minutes,with staining completed in that time.

In certain embodiments, the invention uses flow cytometry sorters ormicrofluidic devices that are identical to or materially similar to flowsorters used to sort live sperm for total DNA content in industrial sexselection. In certain embodiments of the invention, the flow cytometeror microfluidic device utilizes an event rate between 50 to 500 eventsper second, 5,000 to 30,000 events per second, or less than 30,000events per second and sort rates of about 10-35% of the event rate. Incertain embodiments, where sperm are not sorted before analysis, theanalysis rates may be as low as 2-10% of the event rate.

For those embodiments encompassing parse sorting, the inventioncomprises sorting into two or more subpopulations, centered around themean and flanked around the center, where the percentage of parsedcenter cells or nuclei (sort region percentage) is between 25-60% oftotal sperm cells or sperm nuclei, and the percentage of parsed flankedcells or nuclei is the complement/balance of cells or nuclei remainingand in some cases further dividing the flanking sort region to separatethe high-total-DNA content sperm or sperm nuclei on one side of theflank from the low-total-DNA content sperm or sperm nuclei on the otherside of the flank. Alternate embodiments that sort into more than twosubpopulations or require the combinatorial blending of parse samplesare contemplated for use in special situations.

Certain embodiments of the invention encompass extended sorting times toprovide larger quantities of parse sorted samples that may then beanalyzed by microstructural DNA methods such as genotyping or sequencingare anticipated.

In certain embodiments, the invention encompasses the separate analysisof the two or more subpopulations of parse sorted sperm or sperm nuclei(generally corresponding to center and flanking subpopulations). Any ofthe above methods can be applied to these separated subpopulations. Forexample, one may entrain the sperm cells or sperm nuclei, expose them toelectromagnetic radiation, determine a forward fluorescencecharacteristic and a side fluorescence characteristic, gate theindividual events based on the forward fluorescence characteristic andside fluorescence characteristic with one or more criterion and generatea univariate plot based on forward fluorescence intensity to determinethe number of peaks or the peak to valley ratio. Alternatively, a parsesorted sperm cell or nuclei subpopulation can be reprocessed using thelogical gating method or parse sorting method. Alternatively, the parsesorted sperm cells or sperm nuclei may be analyzed by micro-analysis ofDNA such as gene chip analysis or next-generation sequencing (NGS).

In certain embodiments, it is contemplated that univariate plots (e.g.,graphical histograms) are visually inspected for qualitative comparisonof native and parse sorted samples. In other embodiments of theinvention, it is contemplated that flow cytometry data from TRACE filesor related FCS files is mathematically analyzed for quantitativecomparison of native and parse sorted samples. Although the value ofmathematical analysis of flow cytometry data from TRACE files or relatedFCS files may facilitate superior comparative quantification of andidentity of related samples, embodiments of the invention that foregomathematical data analysis (e.g., using only the human eye) may be moresensitive than existing karyotype methods.

Sperm Collection

It is contemplated that intact viable bovine, porcine, equine, ovine,cervine, murine or other mammalian sperm, may be collected for use withthe invention. Various methods of collection of viable sperm cells areknown and include, for example, the gloved-hand method, use of anartificial vagina, and electro-ejaculation. As an example, a bovinesperm sample, typically containing about 0.5 to about 10 billion spermper milliliter, may be collected directly from the source mammal, orfrom more than one source mammal of the same species, into a vesselcontaining an extender to form a sperm cell composition. An extender mayoptionally comprise one or more antioxidants, which may be present asconstituents of the extender prior to contacting with the sperm, orwhich may be added to the sperm cell composition, each antioxidant inthe concentration range of 0.01 mg/ml to 5 mg/ml.

An aggregation reducing compound, or anti-aggregation compound, may alsobe added to the sperm cell composition to prevent aggregation of sperm.Examples of aggregation reducing compounds suitable for use in theinvention include but are not limited to egg yolk, iodixanol, lecithin,bovine serum albumin, gelatin, collagen or hydrolyzed collagen,macromolecules such as arabinogalactan, and chemically definedpolyethylene or polypropylene glycols.

Staining Sperm Cells for Use in the Invention

A process of staining sperm for flow cytometric analysis typicallycomprises the formation of a staining solution containing sperm cellsand a dye, sometimes referred to as a label. A media or extender may becontacted with sperm cells to form a sperm composition, and then thesperm composition contacted with a DNA selective dye to form a stainingsolution. Alternatively, a DNA selective dye may be added to a media oran extender to form a staining solution, with sperm subsequently addedto the staining solution.

The sperm to be stained may be a neat semen (i.e., raw ejaculate), oralternatively, a sperm-containing semen derivative obtained bycentrifugation or the use of other means to separate semen intofractions.

The pH of the staining solution may be maintained at any of a range ofpHs; typically this will be in the range of about 5.0 to about 9.0, orin the range of 5.5 to 7.8. The staining solution may be maintained at aslightly acid pH, i.e., from about 5.0 to about 7.0. Typically, the pHis from about 6.0 to about 7.0; from about 6.0 to about 6.5; about 6.2,about 6.5; about 6.6; about 6.7; about 6.8; about 6.9; or about 7.0.Alternatively, the staining solution may be maintained at a slightlybasic pH, i.e., from about 7.0 to about 9.0. Typically, the pH is about7.0 to about 8.0; about 7.0 to about 7.5; about 7.0; about 7.1; about7.2; about 7.3; about 7.35; about 7.4; or about 7.5.

The staining solution may be formed by using one or more UV or visiblelight excitable, DNA selective dyes as previously described in U.S. Pat.No. 5,135,759 and WO 02/41906, the contents of each of which are herebyincorporated herein by reference. Exemplary UV light excitable,selective dyes include Hoechst 33342 and Hoechst 33258.

The concentration of the DNA selective or of any other type of dye inthe staining solution is a function of a range of variables whichinclude the permeability of the cells to the selected dye, thetemperature of the staining solution, the amount of time allowed forstaining to occur, the concentration of sperm, and the degree ofenrichment desired in the subsequent sorting or enrichment step. Ingeneral, the dye concentration is preferably sufficient to achieve thedesired degree of staining in a reasonably short period of time. Forexample, the concentration of Hoechst 33342 in the staining solutionwill generally be between about 0.1 μM and about 1.0M; from about 0.1 μMto about 1000 μM; from about 100 μM to about 500 μM; from about 200 μMto about 500 μM; or from about 300 μM to about 450 μM. Accordingly,under one set of staining conditions, the concentration of Hoechst 33342is about 350 μM. Under another set of staining conditions, theconcentration of Hoechst 33342 is about 400 μM. Under still another setof staining conditions the concentration is about 450 μM. In someembodiments the concentration of Hoechst 33342 is between about 250 andabout 500 picomoles of Hoechst per 1 million sperm.

Once formed, the staining solution may be maintained at any of a rangeof temperatures; typically, this will be within a range of about 4° C.to about 50° C., but can also be frozen (i.e., cryopreserved). Forexample, the staining solution may be maintained at a relatively lowtemperature, i.e., a temperature of about 4° C. to about 30° C.; in thisembodiment, the temperature is about 20° C. to about 30° C.; from about25° C. to about 30° C.; or about 28° C. Alternatively, the stainingsolution may be maintained within an intermediate temperature range,i.e., a temperature of about 30° C. to about 39° C.; in this embodiment,the temperature is at about 34° C. to about 39° C.; about 35° C.; orabout 37° C. In addition, the staining solution may be maintained withina relatively high temperature range, i.e., a temperature of about 40° C.to about 50° C.; in this embodiment, the temperature is from about 41°C. to about 49° C.; from about 41° C. to about 45° C.; from about 41° C.to about 43° C.; or about 41° C. Selection of a preferred temperaturegenerally depends upon a range of variables, including for example, thepermeability of the cells to the dye(s) being used, the concentration ofthe dye(s) in the staining solution, the amount of time the cells willbe maintained in the staining solution, and the degree of enrichmentdesired in the sorting or enrichment step.

Uptake of dye by the sperm in the staining solution is allowed tocontinue for a period of time sufficient to obtain the desired degree ofDNA staining. That period is typically a period sufficient for the dyeto bind to the DNA of the sperm such that X and Y chromosome-bearingsperm can be distinguished from one another based upon the differing andmeasurable fluorescence intensity between the two. Generally, this willbe no more than about 24 hours; no more than about 30 hours; no morethan about 10 hours; no more than about 2 hours; no more than about 90minutes; no more than about 60 minutes; or from about 5 minutes to about60 minutes. In a particular embodiment, the period is about 30 minutesor about 55 minutes. In another embodiment, the period is less than 5minutes, less than 4 minutes, less than 3 minutes, about 2 minutes, orless than 2 minutes.

Creating Stained Sperm Nuclei for Use in the Invention

One aspect of the invention comprises a staining media for makingstained sperm nuclei. The stained sperm nuclei can in turn be used inthe methods disclosed herein for detecting the presence or absence of achromosomal aberration instead of using intact sperm cells.

In one embodiment, the staining media comprises a buffer, aDNA-selective dye and an aggregation-reducing compound. Any suitablebuffer in the art, such as TRIS citrate, sodium citrate, sodiumbicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, and combinationsthereof, can be used.

Any DNA selective dye known in the art can be used, including but notlimited to Hoechst 33342. In other embodiments, the staining media maybe formed by using one or more UV or visible light excitable,DNA-selective dyes as previously described in U.S. Pat. No. 5,135,759and WO 02/41906, the contents of each of which are hereby incorporatedby reference. Exemplary UV light excitable, selective dyes includeHoechst 33342 and Hoechst 33258.

Additionally, an aggregation-reducing compound may be added to preventaggregation of sperm cells or sperm nuclei, as well as aggregation ofmidpieces and tails. Examples of aggregation-reducing compounds suitablefor use in the invention include but are not limited to egg yolk,iodixanol, lecithin, bovine serum albumin, gelatin, collagen orhydrolyzed collagen, macromolecules such as arabinogalactan, andchemically defined polyethylene or polypropylene glycols. In aparticular embodiment of the invention, the staining media can comprise0.4% or more egg yolk. In another embodiment, the staining media cancomprise between 1-30%, 1-20%, 1-15%, 1-10%, 1-5%, 1-3%, 1-2%, or0.2-1%, egg yolk.

The staining media, or its separate components, can then be combinedwith a sperm cell sample to create a sperm cell mixture. The sperm cellmixture is then sonicated in order to remove midpieces and tails fromthe sperm heads to create sperm nuclei, and to facilitate staining ofthe DNA within the sperm nuclei. In a particular embodiment of theinvention, the sperm cell mixture is sonicated at a sufficientamplitude, frequency or duration to raise the temperature of the spermcell mixture to more than 30, 40, 50, 60 or 70° C. in order tofacilitate staining. In a particular embodiment, the temperature of thesperm cell mixture is raised to more than 50, 60 or 70° C. duringsonication. In another embodiment, the temperature of the sperm cellmixture is raised to at least approximately 60° C. during sonication. Inother embodiments, the target temperature during sonication can be 45°C., or 70° C., or can be in the range between 30−70° C., or 55-65° C. Inanother embodiment, sonication can be carried out on the sperm cellmixture for a particular duration to facilitate staining of sperm nucleiDNA. In one embodiment, the sperm cell mixture can be sonicated forgreater than 1, 2, 3, 4 or 5 minutes. In another embodiment, the spermcell mixture can be sonicated for a total of 1-5 minutes or in an evenmore particular embodiment, approximately 2-3 minutes. In a furtherembodiment of the invention, once the sperm cell mixture is sonicated,the tails and midpieces are removed from the mixture via any knownmethod know in the art, including but not limited to filtration orcentrifugation. Any suitable sonicator can be used to make the spermnuclei. In a particular embodiment, a sonicator with a 20 mhz frequencycan be used to make the sperm nuclei, such as Fisher Scientific ModelFB120. In a more particular embodiment, the sonicator is set to anamplitude of 70%. Finally, one can check whether sonication wassuccessful by examining the sonicated sperm cell mixture bymicroscope—the sonicated sperm cell mixture should substantiallycomprise sperm heads, with midpieces and tails removed and should besubstantially be free of intact sperm cells.

In a particular embodiment, the staining media is made by combining 98.0ml of TRIS-based media, comprising 2% egg yolk, with 2.0 ml of Hoechst33342 (8.1 mM of Hoechst 33342) to yield a final concentration ofHoechst 33342 of 160 μM in the staining media. 1.5 ml of this stainingmedia is then combined with a sperm cell sample of 400 million sperm(extended or raw ejaculate) to create a sperm cell mixture. The spermcell mixture is then sonicated to for about 2 minutes or until itreaches a temperature of about 60° C. This sonication step acts toremove the midpieces and tails from the sperm cells to create spermnuclei (i.e., sperm heads devoid of midpieces and tails) and tofacilitate entry and binding of the DNA-selective dye (in this case,Hoechst 33342) to the DNA within the sperm nuclei.

Use of an Internal Standard with the Invention

In some embodiments, it is contemplated that an internal standard ismixed with the stained sperm cells or stained sperm nuclei beforeanalysis or mixed with each of the parsed sorted subpopulations prior toanalysis. The internal standard may be created from sperm of a differentspecies from the parse sorted subpopulation or the sperm of interestthat is to be analyzed. The internal standard can comprise sperm nuclei,in which case, the internal standard can be made in accordance with theabove disclosure regarding making stained sperm nuclei.

The internal standard is combined with a sperm cell or nuclei sample ora parse sorted sperm or nuclei subpopulation prior to flow cytometricanalysis. One aspect of the invention involves determining the presenceor the absence of a DNA aberration based on a comparison of histogramsof fluorescence intensity of the native (i.e., unsorted) sperm cells orsperm nuclei, or parse sorted subpopulations, with the internalstandard. Generally, when the histograms of the native or parse sortedsubpopulations have a peak to valley ratio that is less than the peak tovalley ratio of the internal standard, a DNA aberration is present.Generally, the Gaussian distribution of modes in the internal standardDNA from the sperm cells or sperm nuclei of the second mammalian speciesand the Gaussian distribution of modes in the normal sample from DNA ofthe reference species represent a combination of standards which canthen be compared to the Gaussian distribution of unknown samples toidentify a DNA aberration.

In one particular embodiment, the internal standard is created using astaining media made by combining 49.0 ml of TRIS-based media comprising20% egg yolk with 432 μl of Hoechst 33342 stain. 1.66 ml of sperm orsperm nuclei (at a concentration of 200 million/ml) are then added tothe staining media. This mixture can then be sonicated to create stainedsperm nuclei.

Detecting the Presence or Absence of DNA Aberrations

One aspect of the invention comprises analyzing stained sperm cells orsperm nuclei via flow cytometry in order to detect the presence orabsence of DNA aberrations, such as chromosomal translocations. As notedabove, in certain embodiments of the invention, sperm cells or spermnuclei analyzed by flow cytometry are also optionally sorted based onfalling within a center or flanking sort region in the context of theparse sorting method for detection of DNA aberrations. Commonly used andwell known sperm cell analysis and sorting methods via flow cytometryare exemplified by and described in U.S. Pat. Nos. 5,135,759, 5,985,216,6,071,689, 6,149,867, and 6,263,745; International Patent PublicationsWO 99/33956 and WO 01/37655; and U.S. patent application Ser. No.10/812,351 (corresponding International Patent Publication WO2004/088283), the content of each of which is hereby incorporated hereinby reference.

In certain embodiments of the invention, analysis of sperm cells may beaccomplished using any process or device known in the art for cellanalysis including but not limited to use of a flow cytometer (includingthe use of a microfluidic chip), and optionally encompasses techniquesfor physically separating sperm from each other, as with droplet sortingand fluid switching sorting, and techniques in which sperm bearing anundesired characteristics are killed, immobilized, or otherwise renderedinfertile, such as by use of laser ablation/photo-damage techniques.Based on the fluorescence emitted by a DNA selective dye upon exposureto a light source such as a high intensity laser beam, a flow cytometer(including a microfluidic device) is able to measure or quantify theamount of DNA present in each cell stained with the DNA selective dye.

A sperm cell or nuclei sample to by analyzed via a flow cytometer(including a microfluidic device) is contained in a sample fluid. Asheath fluid is generally used in a flow cytometer or microfluidicdevice to hydrodynamically focus, entrain or orient sperm or nuclei inthe sample fluid. Generally, the sheath fluid is introduced into anozzle of a flow cytometer or into a microfluidic device usingpressurized gas or by a syringe pump. The pressurized gas is often highquality compressed air. In certain embodiments of the invention, astream containing sperm cells or nuclei to be analyzed may be comprisedof a sample fluid and a sheath fluid, or a sample fluid alone.Optionally, the sample fluid or sheath fluid may also contain anadditive, such as, one or more antioxidants, an antibiotic or a growthfactor, as discussed above with respect to sperm sample collection. Eachof these additives may be added to either fluid in accordance therewith.

FIG. 2 illustrates, in schematic form, part of a flow cytometer used toanalyze and then sort a sperm or nuclei composition to form one or moresubpopulations, the flow cytometer being generally referenced as 10. Theflow cytometer 10 of FIG. 2 can be programmed by an operator to generatetwo charged droplet streams, one containing cells or nuclei within acenter sort region charged positively 12, for example, one containingcells or nuclei within a flanking sort region charged negatively 13 forexample, while an uncharged undeflected stream of indeterminate orundesired cells or nuclei 14 simply goes to waste, each stream collectedin receptacles 28, 29, and 30, respectively.

Initially, a stream of sperm cells or nuclei under pressure, isdeposited into the nozzle 15 from the sperm cell or nuclei source 11 ina manner such that they are able to be coaxially surrounded by a sheathfluid supplied to the nozzle 15 under pressure from a sheath fluidsource 16. An oscillator 17 which may be present can be very preciselycontrolled via an oscillator control mechanism 18, creating pressurewaves within the nozzle 15 which are transmitted to the coaxiallysurrounded sperm cell or nuclei stream as it leaves the nozzle orifice19. As a result, the exiting coaxially surrounded sperm cell or nucleistream 20 could eventually and regularly form droplets 21.

The charging of the respective droplet streams is made possible by thecell sensing system 22 which includes a laser 23 which illuminates thenozzle exiting stream 20, and the light emission of the fluorescingstream is detected by a sensor 24. The information received by thesensor 24 is fed to a sorter discrimination system 25 which very rapidlymakes the decision as to whether to charge a forming droplet and if sowhich charge to provide the forming drop and then charges the droplet 21accordingly.

A characteristic of X chromosome bearing sperm cells or nuclei is thatthey absorb more fluorochrome dye than Y chromosome bearing sperm cellsor nuclei because of the presence of more DNA, and as such, the amountof light emitted by the laser excited absorbed dye in the X chromosomebearing sperm cell or nuclei differs from that of the Y chromosomebearing sperm cells or nuclei. One of the difficulties in accuratequantification of sperm DNA using fluorescence is the geometry of thesperm head, which is shaped like a paddle in most species. Generally,the intensity of fluorescence is lowest when the flat face of the spermis oriented toward a fluorescence detector. This flat orientationactually results in the most accurate measure of DNA content within acell and thus, in sex sorting applications, the best discriminationbetween X and Y chromosome bearing sperm subpopulations. It is thereforedesirable that only properly oriented sperm or sperm nuclei areconsidered in determining the presence or absence of a chromosomalaberrations. There are many techniques known in the art used to orientsperm using various forces generated by the flow cytometer and/ormicrofluidic device, all of which are contemplated for use with theinvention. One way in which orientation can be accomplished in a flowcytometer is by using an orienting nozzle such as described in U.S. Pat.No. 6,357,307, which is hereby incorporated by reference in itsentirety. In one embodiment of the invention, two detectors are used fordetecting fluorescence emitted by sperm cells or nuclei. One of thedetectors is oriented at 0° relative to the laser beam or other sourceof electromagnetic radiation and is used to measure forwardfluorescence, which corresponds to cell DNA content. The second detectoris oriented 90° relative to the laser beam and is used to measure sidefluorescence, which corresponds to the orientation of the sperm cell ornuclei. Since the fluorescence signal is highest for sperm cells ornuclei oriented with their paddle edge toward the side fluorescencedetector, only the sperm cells or nuclei that emit peak fluorescence tothe side fluorescence detector are considered oriented by the flowcytometer.

The charged or uncharged droplet streams pass between a pair ofelectrostatically charged plates 26, which cause them to be deflectedeither one way or the other or not at all depending on their charge intorespective collection vessels 28 and 29 to form a subpopulation of spermcells or sperm nuclei that fell within the center sort region and asubpopulation of cells or nuclei that fell within the flanking sortregion, respectively. The uncharged non-deflected sub-population streamcontaining undesired or indeterminate cells or nuclei go to the wastecontainer 30.

Turning now to FIG. 3, an alternative particle sorting instrument orflow cytometer is partially illustrated in the form of a microfluidicchip (60). The microfluidic chip (60) may include a sample inlet (62)for introducing sample containing particles or cells into a fluidchamber (64) and through an inspection zone (66). Sample introducedthrough the sample inlet (62) may be insulated from interior channelwalls and/or hydrodynamically focused with a sheath fluid introducedthrough a sheath inlet (68). Sample may be interrogated at theinspection zone (66) with an electromagnetic radiation source (notshown), such as a laser, arc lamp, or other source of electromagneticelectricity. Resulting emitted or reflected light may be detected by asensor (not shown) and analyzed with an analyzer (not shown). Each ofthe sheath pressure, sample pressure, sheath flow rate, and sample flowrate in the microfluidic chip may be manipulated in a manner similar toa jet-in-air flow cytometer, by either automatic adjustments performedby the execution of written instructions in the analyzer or by manualadjustments performed by an operator.

In certain embodiments of the invention, once inspected, particles orcells in the fluid chamber (64) may be mechanically diverted from afirst flow path (70) to a second flow path (72) with a separator (74),for altering fluid pressure or diverting fluid flow. The particles orcells may also be permitted to continue flowing along the first flowpath (70) for collection. The illustrated separator (74) comprises amembrane which, when depressed, may divert particles into the secondflow path (72). Other mechanical or electro-mechanical switching devicessuch as transducers and switches may also be used to divert particleflow.

Flow cytometry data analysis is based on the principle of gating.Typically, gates and regions are created around populations of cellswith common characteristics. In the context of the invention, thesecharacteristics can include forward fluorescence and side fluorescence.

Generally, the first step in gating when flow cytometrically analyzingsperm cell or sperm nuclei to determine the presence or absence of a DNAaberration is distinguishing populations of sperm cells or sperm nucleibased on their forward and side fluorescence properties. As noted above,forward and side fluorescence provide an estimate of the DNA content ofthe cells or nuclei and their orientation, respectively. Unorientedsperm cells or nuclei will generate events having a lower level of sidefluorescence, as noted above, and are not resolvable or are lowresolution. These events can be removed by gating on the population ofinterest only (i.e., oriented sperm cells or nuclei).

Gates can be applied to density plots or contour maps to exclude certainpopulations (e.g. unoriented sperm cell or nuclei) or to positivelyselect populations for further analysis, processing or examination.Using analytical software, measurements and statistics can be obtainedfor various parameters in addition to the number of cells or nuclei andpercentage of cells or nuclei within a gate. This can include suchmeasurements as median and mean fluorescence intensity.

Generally, two-parameter density plots (i.e., bivariate plots) displaytwo measurement parameters, one on the x-axis and one on the y-axis andthe events as a density (or dot) plot. The parameters can includeforward florescence intensity, side fluorescence intensity and anintegral of forward florescence intensity.

FIG. 4 illustrates a univariate plot in the form of a histogram that maybe produced by the analyzer (36) and generated into a graphicalpresentation for an operator. The data illustrated in FIG. 4 mayrepresent the number of occurrences of peak signal intensities from theside or forward fluoresce within a certain period. In the case of spermcells or sperm nuclei, X chromosome bearing sperm cells or nuclei and Ychromosome bearing sperm cells or nuclei tend to have peak intensitiesthat vary by between 2 and 5%, depending on the species, and thisdifference is reflected in the bimodal distribution of peak intensities.Because X chromosome bearing sperm cells or nuclei and Y chromosomebearing sperm cells or nuclei tend to have differing fluorescencevalues, each of the peaks represents either X chromosome bearing spermcells or nuclei or Y chromosome bearing sperm cells or nuclei. FIG. 4further illustrates the concept of the peak to valley ratio, which isderived from a relative intensity measurement at the lowest pointbetween the two groups, the valley, which may be considered a value V,and a second relative intensity measurement at the peak or peaks of thehistogram at P.

Example 1

Frozen thawed sperm cell samples of seven different boars werecentrifuged and then gently resuspended in TRIS A, an isotonic saltsolution comprising a TRIS CITRATE buffer with FRUCTOSE and 20% v/vclarified Egg Yolk pH 6.80 and 300 mOsm. A 3-5 ml sample of suspendedsperm cells (concentrations in the range of 200-300 million sperm permilliliter) was combined with Hoechst 333342 in ratio of 500-700nanomoles of stain per 1 million sperm. The mixture was sonicated for2-5 minutes until the temperature of the mixture reached 60° C., therebycreating stained sperm nuclei.

In the parse sorting method, one sort region is used to collect cellsnear the center (center sort region) while the other sort region is usedto collect the remaining cells adjacent to the center sort region onboth sides (flanking sort region). The combined percentages of both sortregions was typically about 90% of the cells. Generally, the percentagein the center sort region was somewhat less than the percentage in theflanking gate. The event rate during parse sorting of the native samplewere typically in the range of 10,000-25,000 cells per second. Theactual event rate chosen for each sample is determined by the operatorand may change during the sorting.

For each of the seven boars exemplified here, the stained sperm nucleisamples were flow cytometrically sorted for the center sort region andflanking sort region into a collection tube. The sorted cells werecentrifuged and decanted to remove most of the fluid. The cells wereresuspended in approximately 300 microliters of the same buffer used forpre-sort staining and sonicating supplemented with about 100 nanomolesof Hoechst 33342 per million sperm and sonicated for 60 seconds beforeanalysis. Since the purpose of the analysis is to have high qualityresolution, and since slower event rates give higher quality resolutiondue to much smaller core stream diameters, event rates for analysis weretypically 50-200 cells per second. The actual event rate chosen for eachsample is determined by the operator and may change during the analysis.

Depending upon whether the boars were normal or had a DNA aberration,the number of peaks in the monovariate histogram were between 2 and 6and the quality (peak to valley ratios) and peak clarity (co-efficientof variation) in the samples varied. Although the different “nativesamples” had different numbers of peaks, the distribution of peaks wastypically symmetric around a central point. In normal boars showing onlytwo peaks (X chromosome and Y chromosome bearing sperm population) thecenter was between those two peaks. In some boars with DNA aberrationsthere only appeared one poor quality peak and in some abnormal boarsthere appeared the typical X and Y peaks near the center as well asadditional clearly defined peaks outside the two central peaks.

FIGS. 5 through 11 (each Figure corresponding to one of the seven boars,respectively) encompass 5 different categories of stable chromosomaltranslocation in found in boars, and the effect the size of translocatedDNA in each of those categories has on the distribution of peaks (modes)in monovariate histograms of the sperm populations when the total DNAcontent is measured by the parse-sorted method. In each figure, theNATIVE histogram represents the data that was used for sorting(parse-sorted method). The PARSED CENTER histogram represents the datafrom sperm populations that were sorted by the CENTER SORT REGION andreanalyzed on the sorter. The PARSED FLANK histogram represents the datafrom sperm populations that were sorted by the FLANKING SORT REGION andreanalyzed on the sorter. The labels representing the 6 peaks (modes)are:

-   -   Y=Sperm cells or sperm nuclei that comprise a completely normal        chromosome content (normal autosomes) and a Y chromosome. Sperm        or sperm nuclei that comprise the short translocation (deleted)        and long translocation (insert) in combination with other normal        autosomes and a Y chromosome. NOTE: This designated mode        contains two genotypes (two different types of chromosomal        combinations) but the same amount of total DNA.    -   X=Sperm cells or sperm nuclei that comprise a completely normal        chromosome content (normal autosomes) and an X chromosome.

Sperm cells or sperm nuclei that comprise the short translocation(deleted) and long translocation (insert) in combination with othernormal autosomes and an X chromosome. NOTE: This designated modecontains two genotypes (two different types of chromosomal combinations)but the same amount of total DNA.

-   -   YS=Sperm cells or sperm nuclei that contain the short        translocation (deletion) with all other normal autosomes and a Y        chromosome.    -   XS=Sperm cells or sperm nuclei that contain the short        translocation (deletion) with all other normal autosomes and an        X chromosome.    -   YL=Sperm cells or sperm nuclei that contain the long        translocation (insert) with all other normal autosomes and a Y        chromosome.    -   XL=Sperm cells or sperm nuclei that contain the long        translocation (insert) with all other normal autosomes and an X        chromosome.

Based on a convention comparing the amount of DNA in the stabletranslocation to the amount of DNA difference between X and Ychromosome, there are 4 categories of visible (measurable) aberrant DNAand the category of normal.

FIG. 5 depicts the parse-sorted analysis of a Boar with a Mos t(7-9)translocation that is typical of a Category I Translocation. In thiscase, since the number of base pairs in the translocated element isenough greater than the number of base pairs in the difference betweenan X chromosome and a Y chromosome, not only does the YS mode containless DNA than the Y mode, the XS mode also contains less. Due to that,there are two modes visible below the Y mode. Conversely, there are alsotwo modes larger than the X mode and even in the NATIVE histogram, it ispossible to see 6 separate peaks. In the analysis of Category I PARSEDCENTER and PARSED FLANK subpopulations that have been parse sorted andreanalyzed separately, the PARSED CENTER histogram is similar to aNORMAL XY distribution, but the PARSED FLANK histogram shows all 6 peaksvery clearly. In this category, the aberrant DNA structure of thetranslocation can already be seen in the NATIVE histogram and is moreclearly confirmed in the reanalyzed samples.

FIG. 6 depicts the parse-sorted analysis of a Boar with an RCP (3-6)2translocation that is typical of a Category II Translocation. In thiscase, since the number of base pairs in the translocated element isabout the same as the number of base pairs in the difference between anX chromosome and a Y chromosome, the XS mode tends to overlap well withthe Y mode and the YL mode tends to overlap well with the X mode. Inthis case, both the NATIVE and the PARSED FLANK histograms show 4 peaks(modes). In this category, the aberrant DNA structure of thetranslocation can already be seen in the NATIVE histogram and is moreclearly confirmed in the reanalyzed samples.

FIG. 7 depicts the parse-sorted analysis of a Boar with an RCP (2q13,15q24) translocation that is typical of a Category III Translocation. Inthis case, since the number of base pairs in the translocated element issomewhat less than the number of base pairs in the difference between anX chromosome and a Y chromosome, the 4-6 peaks are poorly separated inthe NATIVE histogram, but more clearly separated in the PARSED FLANKhistogram. The PARSED CENTER histogram may still show two peaks (modes)that significantly overlap, or even show as one peak. A distinct featureof the Category III Translocation is that the PARSED FLANK histogramshows 4 peaks (modes).

FIG. 8 depicts the parse-sorted analysis of a Boar with an RCP (5-12)translocation that is typical of a Category IV Translocation. In thiscase, since the number of base pairs in the translocated element is verysmall, the X modes (XS, X, XL) are all overlapping in the NATIVEhistogram, while the Y modes (YS, Y, YL) are also overlapping in theNATIVE histogram and the NATIVE histogram looks similar to the NATIVEhistogram of a NORMAL distribution (a boar with no large aberrant DNA).The Category IV Translocation still shows significant differences in thePARSED CENTER and PARSED FLANK analysis, but if the DNA element is verysmall, the only difference may be different CV (or PVR) between them.

FIG. 9 depicts the parse-sorted analysis of a boar with an RCP (5-12)translocation that is typical of a Category IV Translocation where thereanalysis of the PARSED CENTER and PARSED FLANK populations is donewith normal sperm nuclei from cattle (bovine, Jersey breeds) included inthe analysis. The bovine sperm nuclei contain more total DNA than theboar DNA, so the bovine sperm nuclei create a typical two-mode splitseen in sex sorting but shifted to the right. Since both types of spermnuclei are being analyzed together, the quality of the split (separationof modes) in the bovine nuclei is used to optimize the analysis andprove to the operator that the best possible separation has beencreated. That means that the best possible separation has also beencreated in the boar sperm nuclei test sample.

FIG. 10 depicts the parse-sorted analysis of a Boar with NORMAL DNAcontent. In this case, the designation of NORMAL has been created by theobservation that the boar, when analyzed by Karyotype method, shows norecognizable abnormality in chromosome sizes. In this case, the NATIVE,PARSE CENTER and PARSE FLANK analysis all show 2 distinct peaks. Thesomewhat improved separations in the PARSE CENTER and PARSE FLANK arecreated because the reanalysis runs at a slow Event Rate (cell analysisrate) which typically gives better separation.

FIG. 11 depicts the parse-sorted analysis of a boar with NORMAL DNAcontent where the reanalysis includes bovine nuclei as described in FIG.9. The parse-sorted method on NORMAL samples establishes a threshold ofNORMAL vs false positive (falsely described as abnormal when there is noaberrant DNA content) vs false negative (falsely described as normalwhen there is an aberrant DNA content)

Example 2

STAINING/SORTING MEDIA (SSM) used in Example 2—One liter comprises 23.00grams tris(hydroxymethyl)aminomethane (TRIS BASE), 9.56 grams anhydrousfructose, 13.28 grams citric acid monohydrate, 200 mL chicken egg yolk,100 milligrams Hoechst 33342, Trihydrochloride, Trihydrate andapproximately 800 mL of deionized water with a final pH about 6.72.

SORT REANALYSIS MEDIA (SRM) used in Example 2—One liter comprises: 25.88grams tris(hydroxymethyl)aminomethane, 10.75 grams anhydrous fructose,14.94 grams citric acid monohydrate, 100 mL chicken egg yolk, 21.5milligrams Hoechst 33342, Trihydrochloride, Trihydrate and approximately900 ml of deionized water with a final pH about 6.72. If bovine nucleiare included as internal standard, 6.70 billion Bovine Sperm Nuclei(BSN) where BSN are azide stabilized purified sperm heads that have allmidpieces and tail fragments removed and which have been pre-stainedwith Hoechst 33342.

Ejaculate preparation: Frozen ejaculates from 7 boars were thawed at 34°C. and each ejaculate then mixed well using a 5 ml pipette. 7 ml of eachejaculate was placed into a 15 ml conical tube. 7 ml of TrisWS300 wasplaced into each sample and vortexed. Samples were then centrifuged at950 G for 5 minutes, and all supernatant was then decanted leaving thepellet undisturbed. 4 ml of TrisWS300 was added to each sample and thepellet was broken up using a pipette. Each sample was then mixed byvortexing. Cell concentrations were determined using NucleoCounter.

Sample preparation using SSM media: 615 μl of each of the preparedejaculates was then placed in a 5 ml tube (assuming the sampleconcentration was 650 million sperm per ml). Using a P-1000 pipette, 400million sperm cells from each sample were dispensed into a 5 ml sampletube. Using a P-1000 pipette, 1000 μl of SSM (prepared as above) wasdispensed into the same tube. Each sample was then sonicated for 2minutes, taking the samples to approximately 60° C. 500 ul of Tris A wasthen added to each sample and then each sample was sonicated again for˜30 seconds.

Each of the 7 stained sperm nuclei samples was then parse sorted byplacing 5.0 million sperm nuclei from each sample into a 5 ml catch tubeand sorting them on a flow cytometer. A CENTER sort region and a FLANKsort region were placed on the bivariate plot of forward fluorescencepeak vs forward fluorescence integrated. The sorted samples (2 for eachbull) were then centrifuged at 950 G for 5 minutes, and all supernatantwas decanted leaving the pellet undisturbed. 500 μl of SRM (as preparedabove) was then added to each sample and the pellet was broken up with asonicator for 1 minute. Each sorted sample was then analyzed on a flowcytometer at an event rate of between 40-100 events per second.

FIGS. 12 through 18 comprise the 7 examples of using the SSM stainingmedia, sorting of SSM stained boar nuclei with the parse sort method,and reanalysis of the PARSED CENTER and PARSED FLANK populations usingthe SRM media, as indicated above. In all cases, the upper left side ofthe image shows two dot plots: the first shows an oriented gate appliedto SSM stained sperm in a bivariate plot of forward fluorescence (FF) vsside fluorescence (SFF) and the second shows a set of CENTER and FLANKregion polygons that define two separate sort regions on a bivariateplot of forward fluorescence (FF) vs forward fluorescence integrated(FFI, Area). The first monovariate histogram is the NATIVE histogramused for parsed sorting, the second monovariate histogram is the PARSEDCENTER sorted sperm with bovine stained nuclei added and the thirdmonovariate histogram is the PARSED FLANK sorted sperm with bovinestained nuclei added.

FIG. 12 depicts the parse-sorted analysis of a Boar with NORMAL DNAcontent where the reanalysis includes bovine stained nuclei as describedabove for FIG. 9.

FIG. 13 depicts the parse-sorted analysis of a Boar with NORMAL DNAcontent where the reanalysis includes bovine stained nuclei as describedabove for FIG. 9.

FIG. 14 depicts the parse-sorted analysis of a Boar with NORMAL DNAcontent where the reanalysis includes bovine stained nuclei as describedabove for FIG. 9.

FIG. 15 depicts the parse-sorted analysis of a Boar with a Category IVStable Reciprocal Translocation confirmed by Karyotype, with aberrantDNA content where the reanalysis includes bovine stained nuclei asdescribed above for FIG. 9.

FIG. 16 depicts the parse-sorted analysis of a Boar with a Category IStable Reciprocal Translocation confirmed by Karyotype, with aberrantDNA content where the reanalysis includes bovine stained nuclei asdescribed above for FIG. 9.

FIG. 17 depicts the parse-sorted analysis of a Boar with a Category IIIStable Reciprocal Translocation confirmed by Karyotype, with aberrantDNA content where the reanalysis includes bovine stained nuclei asdescribed above for FIG. 9.

FIG. 18 depicts the parse-sorted analysis of a Boar with NORMAL DNAcontent where the reanalysis includes bovine stained nuclei as describedabove for FIG. 9.

Example 3

This example demonstrates the use of logical gating method on boarsperm. After collection, 400 million boar sperm cells from each of foursperm cell samples was mixed with 1 ml of a TRIS-based media comprisingegg yolk and Hoechst 33342. Each sperm cell mixture was then sonicateduntil reaching 60° C. to create stained sperm nuclei. Each stained spermnuclei sample was then analyzed on a flow cytometer having a forward andside fluorescence detector. Events representing oriented sperm nucleiwere first gated on a multivariate plot. A histogram of fluorescenceintensity for all of these gated events was generated (“native”histogram). A second multivariate plot, with forward fluorescenceintensity on the y-axis and an integral of forward fluorescent intensityon the x-axis, was generated based on these gated events. A center gateand a flanking gate were placed on the second multivariate plot.Histograms of fluorescence intensities of events for each of the centergate and the flanking gate were generated. The results are shown inFIGS. 19-22.

FIG. 19 represents the above-referenced histograms (native, center gateand flanking gate) for sperm nuclei obtained from a boar having a stablechromosomal translocation where the quantity of translocated DNA isgreater than the DNA quantity difference between X and Y chromosomes.While the center gate shows two peaks, which is normal, the native andflanking gate histograms show six clearly separated peaks, indicative ofa chromosomal aberration.

FIG. 20 represents the above-referenced histograms (native, center gateand flanking gate) for sperm nuclei obtained from a boar having a stablechromosomal translocation where the quantity of translocated DNA isabout the same as the DNA quantity difference between X and Ychromosomes. While the center gate shows two peaks, which is normal, thehistograms for the native and flanking gate show four clearly separatedpeaks, indicative of a DNA aberration.

FIG. 21 represents the above-referenced histograms (native, center gateand flanking gate) for sperm nuclei obtained from a boar having a stablechromosomal translocation where the quantity of translocated DNA is lessthan the DNA quantity difference between X and Y chromosomes. Thehistograms for the native and the center gate show four poorly separatedpeaks and the flanking gate shows four separated peaks, all indicativeof a DNA aberration.

FIG. 22 represents the above-referenced histograms (native, center andflanking) for sperm nuclei obtained from a boar having a stablechromosomal translocation where the quantity of translocated DNA is muchless than the DNA quantity difference between X and Y chromosomes. Thehistograms for the native and center gate show two poorly separatedpeaks, including having peak to valley ratios of 50% or less, which isindicative of a DNA aberration.

Example 4

After collection, 400 million boar sperm cells from each of eleven spermcell samples was mixed with 1 ml of a TRIS-based media comprising eggyolk and Hoechst 33342. Each sperm cell mixture was then sonicated untilreaching 60° C. to create stained sperm nuclei. Each stained spermnuclei sample was then parse sorted on a flow cytometer having a forwardand side fluorescence detector. Events representing oriented spermnuclei were first gated on a multivariate plot. A histogram offluorescence intensity for all of these gated events was generated(“native” histogram). A second multivariate plot, with forwardfluorescence intensity on the y-axis and an integral of forwardfluorescent intensity on the x-axis, was generated based on these gatedevents. A center sort region and a flanking sort region were provided onthe second multivariate plot. 2 million cells that generated events inthe center sort region and 2 million cells that generated events in theflanking sort region were then separated and collected. Eachsubpopulation was then analyzed separately on a flow cytometer.

For six of the sperm nuclei samples (each sample corresponding to FIGS.23, 29, 30, 31, 32 and 33, respectively), each collected subpopulationwas first centrifuged at 950 g for 5 minutes and decanted. 1 ml ofTRIS-based media comprising egg yolk, Hoechst 33342 and an internalstandard comprising bovine stained sperm nuclei was added to each of thecollected subpopulations from these six sperm nuclei samples. Thesemixtures were then sonicated and separately analyzed on a flowcytometer.

For each of the nine boars evaluated, multivariate plots and histogramsof forward fluorescence intensities were generated for the unsortedsperm nuclei as well as for each of the separated subpopulations thatwere subsequently analyzed. These results are shown in FIGS. 23-33 foreach boar, respectively, and show that the evaluated boars all have DNAaberrations, either by showing more than two separate peaks or peak tovalley ratios of 50% or less on the native, center and flankinghistograms (FIGS. 23-30, 32 and 33), or by showing a peak to valleyratio markedly worse than that generated for the internal standard (see,e.g., FIG. 31). Boars whose results are shown in FIGS. 32 and 33 wereactually karyotyped as normal, which indicates that the invention ismore sensitive in the presence or absence of DNA aberrations thantraditional karyotyping methods.

Example 5

After collection, 400 million bull (bovine) sperm cells from a spermcell sample were mixed with 1 ml of a TRIS-based media comprising eggyolk and Hoechst 33342. The mixture was then sonicated until reaching60° C. to create stained sperm nuclei. The sperm nuclei sample was thenparse sorted on a flow cytometer having a forward and side fluorescencedetector. Events representing oriented sperm nuclei were first gated ona multivariate plot. A histogram of fluorescence intensity for all ofthese gated events was generated (“native” histogram). A secondmultivariate plot, with forward fluorescence intensity on the y-axis andan integral of forward fluorescent intensity on the x-axis, wasgenerated based on these gated events. A center sort region and aflanking sort region were provided on the second multivariate plot.Sperm nuclei that generated events in the center sort region and in theflanking sort region were then separated and collected. Eachsubpopulation was then re-stained with Hoechst 33342 (each 1 millionsperm nuclei subpopulation stained with 20 μl of Hoechst 33342 at aconcentration of 5 mg/ml) and analyzed separately on a flow cytometer.

Multivariate plots and univariate histograms of forward fluorescentintensities were generated for the unsorted sperm nuclei as well as foreach of the separated subpopulations that were subsequently analyzed.These results are shown in FIG. 34, and indicate that the evaluated bullhas a Category IV Translocation.

Example 6

After being collected in an extender, semen samples from 2 boars werebrought to a sperm cell concentration of 200×10⁶ sperm cells/ml in testtubes. 8-10 μl of Hoechst 33342 (at a concentration of 5 mg/ml) wasadded for every ml of extended semen in the samples. Yellow 6 quenchingdye was added at 3 μl for every ml of extended semen in the samples toidentify dead or dying cells. The staining solutions were gently swirledfor 10 seconds and then placed in a water bath at 34.5 to 35° C. for 60minutes. The tubes were inverted every 15 minutes while the samples wereincubating. Each staining solution was then filtered through aCellTrics® filter into properly labeled sample tubes and the stainedsperm cell samples sorted via flow cytometer.

The results for each of the boars' sperm cell samples can be seen inFIGS. 35 and 36, respectively. FIG. 35 indicates that the boar has aCategory IV Translocation due to the bi-modal histogram of forwardfluorescence intensities having a low PVR (only 38%), while FIG. 36indicates that the boar has a Category III Translocation based on thesmall side peaks present on the histogram and low PVR (only 51%).

Example 7

After collection, a bull (bovine)'s semen was extended and stained withHoechst 33342 and a quenching dye using standard techniques whensex-sorting viable sperm. The stained sperm cell sample was then parsesorted on a flow cytometer having a forward and side fluorescencedetector. Events representing oriented sperm nuclei were first gated ona multivariate plot, as can be seen in the upper left portion of FIG.37. A histogram of fluorescence intensity for all of these gated eventswas generated as can also be seen in the middle of FIG. 37, whichindicates that the bull has a Category IV Translocation due to thebi-modal histogram having a low PVR. A second multivariate plot, withforward fluorescence intensity on the y-axis and an integral of forwardfluorescent intensity on the x-axis, was generated based on these gatedevents as can be seen on the lower left portion of FIG. 37. A centersort region and a flanking sort region were provided on the secondmultivariate plot as seen in FIG. 37. 1 million cells that generatedevents in the center sort region and 1 million cells that generatedevents in the flanking sort region were then separated and collected.Each subpopulation was then re-stained with Hoechst 33342 (each 1million sperm nuclei subpopulation stained with 20 μl of Hoechst 33342at a concentration of 5 mg/ml and 20 μl of TRIS-based media with 20% eggyolk), sonicated and then analyzed separately on a flow cytometer.

The resulting univariate histograms of forward fluorescence intensitiesfor each subpopulation are shown in FIGS. 38 and 39 respectively, whichindicate the presence of a Category IV translocation (FIG. 38 shows asingle mode (i.e. the absence of a PVR), while FIG. 39 shows a bi-modalhistogram with a low PVR).

What we claim is:
 1. A method of analyzing fluorescence emissioncharacteristics in sperm cells or sperm nuclei, comprising: entrainingsperm cells or sperm nuclei stained with a DNA selective dye in sheathfluid; exposing the entrained sperm cells or sperm nuclei toelectromagnetic radiation; determining a forward fluorescencecharacteristic and a side fluorescence characteristic of individualevents associated with the exposed sperm cells or sperm nuclei; gatingthe individual events based on the forward fluorescence characteristicand the side fluorescence characteristic with a criterion; anddetermining the presence or absence of a DNA aberration from the gatedindividual events.
 2. The method of claim 1, wherein the DNA selectivedye is Hoechst
 33342. 3. The method of claim 1, further comprising thestep of orienting the entrained sperm cells or sperm nuclei.
 4. Themethod of claim 1, wherein the step of exposing the sperm cells or spermnuclei to electromagnetic radiation comprises exposing the sperm cellsor sperm nuclei to a laser beam with modified beam profile.
 5. Themethod of claim 1, wherein the criterion encompasses a subpopulation oforiented sperm cells or sperm nuclei.
 6. The method of claim 1, whereinthe sperm cells or sperm nuclei comprise sperm cells or sperm nucleifrom a first and a second mammalian species.
 7. The method of claim 1,wherein determining the presence of a DNA aberration from the gatedindividual events comprises detecting more than two peaks or modes, or apeak to valley ratio of 50% or less, 60% or less, 70% or less or 80% orless, on a histogram of fluorescence intensities.
 8. The method of claim6, wherein determining the presence of a DNA aberration from the gatedindividual events comprises detecting a difference between a peak tovalley ratio on a histogram of fluorescence intensities of sperm cellsor sperm nuclei from the first mammalian species and a peak to valleyratio on a histogram of fluorescence intensities of sperm cells or spermnuclei from the second mammalian species.
 9. A method of analyzingfluorescence emission characteristics in sperm cells or sperm nuclei,comprising: entraining sperm cells or sperm nuclei stained with a DNAselective dye in sheath fluid; exposing the entrained sperm cells orsperm nuclei to electromagnetic radiation; determining two or morefluorescence emission characteristics of individual events associatedwith the exposed sperm cells or sperm nuclei; gating the individualevents based on the two or more fluorescence emission characteristicswith a first criterion for further processing; gating the furtherprocessed individual events with a second criterion; and determining thepresence or absence of a DNA aberration from the twice gated individualevents.
 10. The method of claim 9, wherein the first criterionencompasses a first subpopulation of oriented sperm cells or spermnuclei.
 11. The method of claim 10, wherein the second criterionencompasses a second subpopulation of sperm cells or sperm nucleicomprising 25% to 75% of the first subpopulation.
 12. The method ofclaim 11, wherein the step of determining the presence or absence of DNAaberrations in the sperm cells or sperm nuclei from the twice gatedindividual events further comprises evaluating a third subpopulation ofsperm cells or sperm nuclei within the first subpopulation, wherein thethird subpopulation of sperm cells or sperm nuclei excludes the secondsubpopulation of sperm cells or sperm nuclei.
 13. The method of claim10, wherein the second criterion encompasses 25% to 75% of sperm cellsor sperm nuclei around a median value of a fluorescence emissioncharacteristic.
 14. The method of claim 9, wherein the step ofdetermining the presence or absence of a DNA aberration in the spermcells or sperm nuclei from the twice gated individual events furthercomprises determining the quantity of peaks or modes in a twice gatedfluorescence emission characteristic.
 15. The method of claim 9, whereinthe step of determining the presence or absence of a DNA aberration inthe sperm cells or sperm nuclei from the twice gated individual eventsfurther comprises analyzing a coefficient of variation of a fluorescenceemission characteristic associated with the first subpopulation of spermcells or sperm nuclei or the second subpopulation of sperm cells orsperm nuclei.
 16. The method of claim 9, wherein the step of determiningthe presence or absence of a DNA aberration in the sperm cells or spermnuclei from the twice gated individual events further comprisesgenerating a first univariate plot based on a fluorescence emissioncharacteristic of the first subpopulation or generating a secondunivariate plot based on a fluorescence emission characteristic of thesecond subpopulation.
 17. The method of claim 16, wherein the step ofdetermining the presence or absence of a DNA aberration in the spermcells or sperm nuclei from the twice gated individual events furthercomprises analyzing a peak to valley ratio of the first univariate plotor a peak to valley ratio of the second univariate plot.
 18. The methodof claim 9, further comprising the step of separating tails or midpiecesfrom the sperm nuclei by centrifugation.
 19. A method of analyzing afluorescence emission characteristic in sperm cells or sperm nucleicomprising: staining the sperm cells or sperm nuclei with a DNAselective dye; entraining the stained sperm cells or sperm nuclei insheath fluid; exposing the entrained sperm cells or sperm nuclei toelectromagnetic radiation; determining a fluorescence emissioncharacteristic of the exposed sperm cells or sperm nuclei; generating afirst multivariate plot based on the fluorescence emissioncharacteristic; providing a first gate on the first multivariate plot;generating a second multivariate plot based on the first gate; providinga first sort region on the second multivariate plot encompassing a firstsubpopulation of sperm cells or sperm nuclei and a second sort region onthe second multivariate plot encompassing a second subpopulation ofsperm cells or sperm nuclei; separating the first subpopulation of spermcells or sperm nuclei and the second subpopulation of sperm cells orsperm nuclei; generating a first univariate plot based on a fluorescenceemission characteristic of the separated first subpopulation of spermcells or sperm nuclei; generating a second univariate plot based on afluorescence emission characteristic of the separated secondsubpopulation of sperm cells or sperm nuclei.
 20. The method of claim19, further comprising the step of removing tails or midpieces from thesperm nuclei by centrifugation.
 21. The method of claim 20, wherein thefirst subpopulation of sperm cells or sperm nuclei comprises 25% to 75%of the sperm cell or sperm nuclei population.
 22. The method of claim21, wherein the second subpopulation of sperm cells or sperm nucleiexcludes the first subpopulation of sperm cells or sperm nuclei.
 23. Themethod of claim 22, wherein the first sort region encompasses the centerof the second multivariate plot.
 24. The method of claim 21, furthercomprising the step of determining the quantity of peaks or modes of thefirst univariate plot or the second univariate plot, analyzing acoefficient of variation of the first univariate plot or the secondunivariate plot, or analyzing a peak to valley ratio of the firstunivariate plot or the second univariate plot.
 25. A compositioncomprising unsorted sperm nuclei, a DNA-selective dye and an aggregationreducing compound, wherein the composition has been sonicated.
 26. Thecomposition of claim 25, wherein the aggregation reducing compoundcomprises egg yolk, iodixanol, lecithin, bovine serum albumin, gelatin,collagen or hydrolyzed collagen, arabinogalactan, or a chemicallydefined polyethylene or polypropylene glycol.
 27. The composition ofclaim 25, wherein the sperm nuclei comprise sperm nuclei from a firstnon-human mammalian species and sperm nuclei from a second non-humanmammalian species.
 28. A method of analyzing a fluorescence emissioncharacteristic in sperm nuclei comprising: combining a DNA-selective dyeand an aggregation reducing compound with a sperm cell sample to createa sperm cell mixture; sonicating the sperm cell mixture to createstained sperm nuclei; determining a forward fluorescence characteristicand a side fluorescence characteristic of individual events associatedwith the exposed stained sperm nuclei; determining the presence orabsence of a DNA aberration from the individual events.